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Nanocarriers Designed to Deliver Nucleic Acids to Brain

旨在将核酸输送到大脑的纳米载体

基本信息

批准号：
9770565
负责人：
JUAN R SANCHEZ-RAMOS
金额：
$ 35.05万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9770565
关键词：
Applications Grants Behavior Behavioral Blood - brain barrier anatomy Brain Brain Diseases Brain region Caliber Cell Culture Techniques Cells Cerebral cortex Chelating Agents Chitosan Chronic Clinical Corpus striatum structure Culture Media DNA delivery Data Development Diphosphates Dose Drug Delivery Systems Electron Microscopy Extracellular Fluid Fibroblasts Fluorescence Fluorescence Microscopy Formulation Gene Silencing Genes Goals Green Fluorescent Proteins Hour Huntington Disease Image Incubated Infusion procedures Injections Intranasal Administration Intrathecal Space Label Life Magnetic Resonance Imaging Manganese Measures Messenger RNA Metals Methods Microscopy Motor Activity Mouse Cell Line Mus Neurodegenerative Disorders Neurons Nose Nucleic Acids Olfactory Mucosa Operative Surgical Procedures Pathologic Patients Pharmaceutical Preparations Plasmids Play Process Research Proposals Residual state Role Series Signal Transduction Small Interfering RNA System Technology Testing Therapeutic Therapeutic Agents Time Transfection Transgenic Mice Transgenic Organisms Viral Vector Western Blotting base clinically significant comparative crosslink cytotoxicity design ds-DNA exosome experimental study extracellular gene therapy in vivo light scattering mRNA Expression mouse model nanocarrier nanoparticle novel therapeutics nucleic acid-based therapeutics olfactory bulb primary endpoint protein expression public health relevance red fluorescent protein

项目摘要

DESCRIPTION (provided by applicant): Gene therapy of brain diseases is hampered by the requirement for invasive stereotaxic injection into brain, or infusion of therapeutic agents into te intrathecal space. The surgical approach is not optimal for neurodegenerative diseases that require treatments throughout life. This obstacle has given impetus to the design of a new nose-to-brain delivery system for nucleic acids or drugs that cannot pass the blood-brain-barrier. The overall goal of this project is to refine and test a nanocarrier system consisting of chitosan-based, manganese-containing nanoparticles (mNPs) loaded with therapeutic nucleic acids: small interfering RNA (siRNA) and dsDNA. The experiments are designed to assess the mechanisms and extent to which the nanocarriers transport their payload from the olfactory mucosa to olfactory bulb and other brain regions to suppress marker genes in mouse models of rapid onset Huntington's Disease. Specific Aim 1: Testing optimized nanocarriers loaded with siRNA against a marker gene expressed in transgenic "green" mice that constitutively express green fluorescent protein (GFP) in brain neurons. mNPs will be packaged with siRNA directed against GFP. Primary end-points will be a) the extent and time-course of dissemination of mNPs from olfactory bulb to other regions of brain assessed by MRI T1-weighted imaging; b) GFP mRNA and GFP protein expression by comparative quantitiative PCR and Western blot analysis, respectively. Specific Aim 2: Study of gene-silencing in a mouse model of HD. siRNA directed against htt will be loaded into NPs and administered intra-nasally or microinjected directly into striatum in rapid-onset mouse models of Huntington's Disease. Primary-end points will be a) extent and time-course of dissemination of mNPs from olfactory bulb (or from striatum) to other regions of brain assessed by microbore MRI, b) quantitative analysis of htt mRNA expression by quantitative PCR and htt protein expression by Western blot in various brain regions, c) effects on behavior and locomotor activity. Specific Aim 3 a) Iterative optimization of the nanocarrier system in cell cultures expressing a marker gene guided by results from concurrent in vivo experiments in Aim 1 and b) studies of cellular extrusion of exosomes containing mNPs as an hypothesized mechanism for cell-to-cell dissemination. Specific Aim 4: Testing capacity of mNPs to deliver DNA (gene encoding a red fluorescent protein) in vivo. mNPs will be loaded with plasmid dsDNA encoding red fluorescent protein (RFP) and intranasally instilled into normal C57BL6 mice. End-points will be the same as those in Aim 2. Clinical Significance: The ability to dose patients chronically and non-invasively via intra-nasal administration of nanocarriers of gene-silencing agents or other large therapeutic molecules will have a dramatic impact in the therapeutics of brain disorders.

描述（由申请人提供）：脑部疾病的基因治疗受到侵入性立体定位注射到脑部或将治疗剂输注到鞘内空间的要求的阻碍。手术方法对于需要终生治疗的神经退行性疾病不是最佳的。这一障碍推动了设计一种新的鼻-脑递送系统，用于不能通过血脑屏障的核酸或药物。该项目的总体目标是改进和测试由负载有治疗性核酸的基于壳聚糖的含锰纳米颗粒（mNPs）组成的纳米载体系统：小干扰RNA（siRNA）和dsDNA。这些实验旨在评估纳米载体将其有效载荷从嗅粘膜运输到嗅球和其他脑区域以抑制快速发作的亨廷顿病小鼠模型中的标记基因的机制和程度。具体目标1：测试负载有针对转基因“绿色”小鼠中表达的标记基因的siRNA的优化的纳米载体，所述转基因“绿色”小鼠在脑神经元中组成型表达绿色荧光蛋白（GFP）。将用针对GFP的siRNA包装mNP。主要终点将是a）通过MRI T1加权成像评估的mNP从嗅球到脑的其它区域的扩散的程度和时间过程; B）分别通过比较定量PCR和Western印迹分析的GFP mRNA和GFP蛋白表达。具体目标2：HD小鼠模型中基因沉默的研究。将针对htt的siRNA加载到NP中，并鼻内施用或直接显微注射到亨廷顿病的快速发作小鼠模型中的纹状体中。主要终点将是a）通过微孔MRI评估的mNP从嗅球（或从纹状体）向脑的其它区域的扩散的程度和时间过程，B）通过定量PCR定量分析不同脑区域中的htt mRNA表达和通过Western印迹定量分析htt蛋白表达，c）对行为和运动活性的影响。具体目标3 a）迭代优化由来自目标1和B）研究中的同时进行的体内实验的结果指导的表达标记基因的细胞培养物中的纳米载体系统，所述研究是含有mNP的外来体的细胞挤出作为细胞间传播的假设机制。具体目的4：测试mNP在体内递送DNA（编码红色荧光蛋白的基因）的能力。将mNP装载有编码红色荧光蛋白（RFP）的质粒dsDNA，并鼻内滴注到正常C57 BL 6小鼠中。终点将与目标2中的终点相同。临床意义：通过鼻内给药基因沉默剂或其他大治疗分子的纳米载体长期和非侵入性地给药患者的能力将在脑疾病的治疗中产生巨大影响。